This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The "Activation Induced Deaminase" (AID-protein) is potentially involved in RNA editing in human lymphocytes. In search for possible interacting proteins, that could function in the assumed recruitment of AID to specific RNAs, we have employed MALDI-ion trap MS/MS analysis to identify proteins that are co-purified with flag/HA-tagged AID. We have also identified sites of phosphorylation on AID at a site that appears to play an important role in regulating AID activity. Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching. Three manuscripts describing this work has been published: K.M. McBride, A. Gazumyan , E.M. Woo , V.M. Barreto, D. F. Robbiani, B.T. Chait , M.C. Nussenzweig "Regulation of hypermutation by AID phosphorylation" Proc Natl Acad Sci 103 (2006) 8798-803 K.M. McBride, A. Gazumyan, E.M. Woo, T.A. Schwickert, B.T. Chait, M.C. Nussenzweig, "Regulation of class switch recombination and somatic mutation by AID phosphorylation", J Exp Med. 205 (2008) 2585-94. Gazumyan A, Timachova K, Yuen G, Siden E, Di Virgilio M, Woo EM, Chait BT, San-Martin BR, Nussenzweig MC, McBride KM Amino Terminal Phosphorylation of Activation-Induced Cytidine Deaminase Suppresses c-myc-IgH translocation Mol Cell Biol 2010 Dec 6. [Epub ahead of print]